The structural basis for discrimination of DNA substrates by Cre recombinase will be investigated. Cre recombinase promotes a specific crossing-over reaction between two 34 basepair loxP DNA sequences in vitro and in vivo. The Cre-lox system has shown great utility for generating specific chromosomal rearrangements in living cells and organisms. Its greatest limitation is the requirement for introducing lox sequences into the target genomes by relatively inefficient methods. Although lox-related sequences (LRSs) occur in mammalian genomes, they do not function efficiently enough for most applications. If the structural mechanisms for discriminating IoxP sequences from other DNA can be identified, they could be redirected by protein engineering to allow Cre to recognize existing genomic LRSs and non-lox sequences. The large size of the lox sequence, the multimeric active complex, and multi-step reaction allow for several levels of substrate discrimination. To define the sequence requirements for efficient recombination of the loxP site at the nucleotide level, the recombination activity of a systematic set of singly and doubly substituted loxP sequences will be quantitatively assessed. For low activity substrates, the reaction step(s) that is(are) involved in discrimination will be determined by analysis of DNA binding, active complex assembly and catalysis. To understand the structural basis for inactivity of substrates which are bound but do not undergo recombination, X-ray crystal structures of the impaired Cre-variant lox complexes wilt be analyzed for structural differences from the cognate loxP complexes. The role of an eight nucleotide region of LoxP that separates the two Cre binding sites in promoting efficient recombination will be ascertained from reaction intermediate structures incorporating this region. These structures wilt also act as references for comparisons with the mutant complexes. Since the Cre-lox reaction is paradigmatic for site-specific recombination, these results of this work should be applicable to understanding control of specificity by other tyrosine recombinases.